Allergy
Immune responses that are elicited in response to many otherwise innocuous environmental allergens, as well as in response to infections with many parasites, often are associated with high levels of immunoglobulin E (“IgE”) production. The amount of immunoglobulins E/High affinity IgE receptor at the surface of basophils can be informative of the allergic status as well as the fact that patients can be on therapy. It generally is believed these immune responses are promoted by antigen-specific T helper 2 (Th2) cells and that unwanted IgE-associated immune responses (i.e., allergic diseases) are the unfortunate result of the immune system perceiving and responding to otherwise essentially harmless allergens as if they were derived from a parasite.
In the context of allergic diseases, allergen challenge of a sensitized host can result in a range of tissue responses, depending on such factors as the route and dose of allergen challenge, and on whether the allergen challenge represents a single transient exposure, results in the persistence of the allergen, or occurs seasonally (such as hay fever) or in some other repetitive fashion. Tissue responses also may be affected importantly by the genetic background of the host and by diverse nongenetic factors (such as certain concurrent infections), which can modify the host's response to allergen.
The effector phases of IgE-associated immune responses may be described as occurring in three temporal patterns: (i) acute reactions (acute response), which develop within seconds or minutes of allergen exposure; (ii) late-phase reactions (late phase response), which develop within hours of allergen exposure, often after at least some of the effects of the acute reaction have partially diminished; and (iii) chronic allergic inflammation (chronic allergic response), which can persist for days to years.
In the early stages of allergy, a hypersensitivity reaction against an allergen, encountered for the first time, causes a response in Th2 cells, a subset of T cells that produce the cytokine interleukin-4 (“IL-4”). The Th2 cells interact with B cells (lymphocytes that produce antibodies against antigens), and, coupled with the effects of IL-4, stimulate the B cells to begin production and secretion of IgE. The secreted IgE circulates in the blood and binds to the high affinity IgE receptor (“FcεRI”) on the surface of mast cells and basophils, both of which are involved in the acute inflammatory response. At this state, the IgE-coated cells are sensitized to the allergen.
If later exposure to the same allergen occurs, the allergen can bind to the IgE molecules held on the surface of the mast cells or basophils. Cross-linking of the IgE and Fc receptors occurs when more than one IgE-receptor complex interacts with the same allergenic molecule, and activates the sensitized cell. Subsequently, these activated mast cells and basophils undergo the process of degranulation during which they release histamine and other inflammatory chemical mediators, such as cytokines, interleukins and prostaglandins, from their granules into the surrounding tissue causing several systemic effects, such as, for example, but not limited to, vasodilation, mucous secretion, nerve stimulation, and smooth muscle contraction. This may result in rhinorrhea (runny nose), itchiness, dyspnea (difficulty in breathing), or anaphylaxis. Depending on the individual patient, allergen, and mode of introduction, the symptoms may be system-wide (classical anaphylaxis) or localized to particular body systems, such as asthma (localized to the respiratory system) and eczema (localized to the dermis).
After the chemical mediators of the acute response subside, late phase responses may occur. Tissues may become red and swollen due to the migration, initiated by the release of cytokines from mast cells and basophils, of other leukocytes, such as neutrophils, lymphocytes, eosinophils and macrophages, to the initial site. Platelets also may participate. The reaction usually is seen from 2 hours to 24 hours after the original reaction.
Allergic Diseases
Allergic diseases are the group of hypersensitivity disorders that may be (a) associated with the production of specific IgE to environmental allergens and (b) thought to involve, as part of their pathogenesis, IgE-mediated reactions. These reactions are prevalent. Allergic reaction can be IgE independent as well.
Anaphylaxis
Anaphylaxis is an acute, systemic, hypersensitivity response to allergen, which typically involves multiple organ systems and which, if untreated, rapidly can lead to death. The vast majority of anaphylactic or anaphylactoid reactions encountered clinically are due to IgE-dependent reactions to penicillin or other antibiotics, foods, or the venom of stinging insects. Further, anaphylaxis also may be IgE independent. It generally is believed that most, if not all, of the signs and symptoms of IgE-associated anaphylaxis in humans reflect (a) the systemic, FcεRI-dependent activation of mast cells and/or basophils and (b) the end-organ consequences of the release of mediators by these cells. Mild cases of acute systemic allergic reactions may primarily involve the skin, which exhibits widespread areas of increased vascular permeability, erythema, and itching (hives). In more severe cases, greatly increased vascular permeability occurs in multiple organ systems, including the upper airways, leading to laryngeal edema and upper airway obstruction. Further, the rapid loss of intravascular fluid volume, together with other consequences of mediator release in anaphylaxis, such as loss of tone in capacitance vessels and decreased contractility of the heart, leads to hypotension and shock. Breathing also may be impaired by marked narrowing of the lower airways, resulting in a severe case of acute asthma, and there may be pronounced gastrointestinal signs and symptoms, such as nausea and vomiting.
Allergic Rhinitis
Allergic rhinitis (hay fever) is one of the most prevalent allergic diseases. It generally is believed that symptoms, which include sneezing, nasal congestion and itching, and rhinorrhea (runny nose), primarily reflect the IgE-dependent release of mediators by effector cells (mainly mast cells and basophils) in response to aeroallergens. Accordingly, symptoms may be seasonal, correlating with the presence of the offending grass, weed or tree pollens, or mold spores, or year-round (for example, the presence of dust mites or animal dander). Typically, symptoms develop rapidly upon exposure to allergen. Nasal tissues usually exhibit marked infiltration with eosinophils and basophils.
Asthma
Asthma is an airway disease that can be classified physiologically as a variable and partially reversible obstruction to air flow, and pathologically with overdeveloped mucus glands, airway thickening due to scarring and inflammation, and bronchoconstriction (the narrowing of the airways in the lungs due to the tightening of surrounding smooth muscle). Bronchial inflammation also causes narrowing due to edema and swelling caused by an immune response to allergens. In human allergic asthma, it generally is believed that IgE-dependent mast-cell activation importantly contributes to acute allergen-induced bronchoconstriction, and that mast cells can contribute to the airway inflammation associated with this disorder. In humans, the FcεRI can be expressed on several potential effector cells in addition to basophils and mast cells, including monocytes, macrophages, eosinophils, neutrophils and platelets. IgE can directly or indirectly upregulate FcεRI expression on basophils and mast cells, and, by binding to this receptor, prime the cells to release increased amounts of key mediators, such as histamine, IL-4, IL-13, MIP-1α and other cytokines.
Atopic Dermatitis
Atopic dermatitis is an inflammatory, chronically relapsing, non-contagious and pruritic skin disease. The skin of a patient with atopic dermatitis reacts abnormally and easily to irritants, food, and environmental allergens and becomes red, flaky and very itchy. It also becomes vulnerable to surface infections caused by bacteria. The skin on the flexural surfaces of the joints (for example, inner sides of elbows and knees) is the most commonly affected region in humans. Naturally occurring lesions of atopic dermatitis may include T cells, along with eosinophils and their products, although their roles are unclear.
Atopic dermatitis often occurs together with other atopic diseases like hay fever, asthma and conjunctivitis. It is a familial and chronic disease and its symptoms can increase or disappear over time. Atopic dermatitis in older children and adults often is confused with psoriasis. Atopic dermatitis afflicts humans, particularly young children; it is also a well-characterized disease in domestic dogs. There is no cure for atopic eczema, and its causes are not well understood.
Mastocytosis
Mastocytosis refers to a group of disorders characterized by excessive mast cell accumulation in one or multiple tissues. Mastocytosis is subdivided into two groups of disorders: systemic or cutaneous. A subset of of patients with recurrent anaphylaxis, but without mastocytosis, have been reported to carry clonal markers of mast cell disease such as, for example, D816V c-kit mutation. Anaphylaxis can be observed in both cutaneous and systemic mastocytosis.
Eosinophilic Esophagitis
Eosinophilic esophagitis (EoE) is part of a heterogeneous group of eosinophil-associated gastrointestinal disorders that is characterized by high numbers of eosinophils infiltrating into the esophagus. While the incidence of EoE is increasing, precise mechanisms of this disease remain largely unknown, though EoE is associated with allergy. Currently, eosphagogastroduodenoscopy (EGD) and histological examination of esophageal biopsies are required for the diagnosis of EoE, and repeated procedures often are employed for the assessment of response to therapy. Current treatments rely on avoidance of specific food and airborne allergens in atopic patients, anti-inflammatory drugs, such as glucocorticoids, or experimental drugs, such as mepolizumab. The need for less invasive procedures to diagnose and monitor EoE remains.
Angioedema
Angioedema is a self-limited, localized swelling of the skin, which results from extravasation of fluid into interstitial tissues. It affects the skin and mucosal tissues of the face, lips, mouth, and throat, larynx, extremities, and genitalia, often in an asymmetric pattern. Bowel wall involvement presents as a colicky abdominal pain. Angioedema may occur in isolation, accompanied by urticaria, or as a component of anaphylaxis. Mast cell-mediated angioedema is associated with urticaria and/or pruritus in 90 percent of cases. There are many agents, including drugs and allergens, that can result in mast cell-mediated angioedema.
Autoimmune Disorders
The term “autoimmune disorder” as used herein refers to disease, disorders or conditions in which the body's immune system, which normally fights infections and viruses, is misdirected and attacks the body's own normal, healthy tissue. Mast cells are implicated in the pathology associated with the autoimmune disorders rheumatoid arthritis, bullous pemphigoid, and multiple sclerosis. They have been shown to be involved in the recruitment of inflammatory cells to the joints (e.g. rheumatoid arthritis) and skin (e.g. bullous pemphigoid) this activity is dependent on antibodies and complement components.
In the condition autoimmune mast cell release, recurrent episodes of angioedema and urticaria may persist over months to years. Angioedema is present in up to 50 percent of patients with chronic urticaria. In this condition, symptoms occur independently of external triggers. One proposed mechanism is the formation of autoantibodies to either IgE or the IgE receptor on mast cells, which then activate the cells intermittently. Chronic urticaria can be associated with the presence of Anti-Fc(episilon)RI auto antibodies. Patients with autoantibodies have both markedly reduced basophil numbers and basophil histamine release
Monoclonal Gammopathies (Paraproteinemias or Dysproteinemias)
The monoclonal gammopathies (paraproteinemias or dysproteinemias) are a group of disorders characterized by the proliferation of a single clone of plasma cells which produces an immunologically homogeneous protein commonly referred to as a paraprotein or monoclonal protein (M-protein, where the “M” stands for monoclonal). Each serum M-protein consists of two heavy polypeptide chains of the same class designated by a capital letter and a corresponding Greek letters: Gamma (γ) in IgG, Alpha (α) in IgA, Mu (μ) in IgM, Delta (δ) in IgD, Epsilon (ε) in IgE. Basophils in IgE myeloma are characterized by a higher expression of high affinity IgE receptor relative to normal controls.
White Blood Cells
White blood cells (“leukocytes”, “WBCs”) are cells of the immune system that defend the human body against infectious disease and foreign materials. The name “white blood cell” derives from the fact that after centrifugation of a blood sample, the white cells are found in a thin, typically white layer (“buffy coat”) of nucleated cells between the pelleted red blood cells and the blood plasma.
The several different types of WBCs, including neutrophils, eosinophils, basophils, lymphocytes, monocytes, macrophages and dendritic cells, often divided into two subgroups, granulocytes or agranulocytes, based on their appearance by light microscopy.
1. Granulocytes
Granulocytes (polymorphonuclear leukocytes) are leukocytes characterized by the presence of differently staining granules in their cytoplasm when viewed under light microscopy. These granules are membrane-bound enzymes that act primarily in the digestion of endocytosed particles. Granulocytes include basophils, eosinophils and neutrophils.
Basophils
Basophilic granulocytes (basophils) are a small population of peripheral blood leukocytes containing cytoplasmic granules that stain with basophilic (staining readily with a basic dye) dyes. Based on their similar morphology to mast cells, basophils have often been considered (and neglected) as minor and possibly redundant “circulating mast cells.” It has been very difficult for most laboratories to obtain basophils without major contaminating populations, because the percentage of basophils in peripheral blood is low (<1%) and they share physiochemical properties with other blood cells. This lack of satisfactory purification protocols has considerably hampered basophil research and negatively affected the interest in this cell type.
Basophils contain prominent cytoplasmic granules, are major sources of histamine (a vasodilator) and other potent chemical mediators of inflammation, and constitutively express Fc epsilon receptor (FceRI), the high affinity IgE receptor. They typically exhibit a segmented nucleus with marked condensation of nuclear chromatin. As with all granulocytes, basophils develop in the bone marrow, and are released as fully mature cells with a survival span estimated to be 2-3 days.
Basophils express a variety of seven membrane transverse receptors that bind chemotatic factors. Most are members of the CCR family of receptors that bind CC (or the β-family) chemokines. Among those with overlapping binding (predominantly to CCR3) are members of the monocyte chemotactic protein (MCP) family, including MCP-1 (CCL2), MCP-3 (CCL7), MCP-4 (CCL13), RANTES (CCL5), MIP-1α (CCL3), eotaxin-1 (CCL11), and eotaxin-2 (CLL24). Further, basophils have receptors for stromal cell-derived factor (SDF-1; CXCL12), a strongly chemotactic molecule for lymphocytes that is a member of the CXC family that binds to CXCR-4; a receptor with a wide cellular distribution, expressed on the surface of most immature and mature hematopoietic cells types such as, for example, neutrophils, monocytes, T and B cells, dendritic cells, Langerhans cells, and macrophages.
Human basophils also express several cytokine receptors. Among these are receptors that bind to specific interleukins including IL-2, IL-3, IL-4, IL-5, and IL-33. Only IL-3 and IL-33 are thought to mediate significant functional responses. Basophils are but one of two cell types in blood (the other being plasmacytoid dendritic cells (pDCs)) that express IL-3 receptors (CD123) at exceedingly high levels. Although the exact number of receptors remains unknown, studies indicate the expression levels of IL-3 are nearly 2-fold higher than any other cell type. This characteristic has led to use of CD123 expression as a marker to specifically gate on basophils (and pDCs) during flow cytometry analysis.
The high affinity IgE receptor (FcεRI) is thought to be the single most significant activation-linked molecule known on basophils. These receptors are comprised of four subunits: one α, one β, and 2γ chains that form a tetramer structure (αβγ2). Two extracellular domains on the α-subunit allow IgE binding, whereas signaling events are initiated through immunoreceptor tyrosine-based activation motifs (ITAMs) located within intracellular portions of the β-subunits and γ-subunits. In humans, a trimeric form of FcεRI, which lacks the β-subunit (αγ2), also is found on antigen-presenting cells (APCs), including Langerhans cells, monocytes and blood dendritic cells. Mast cells, eosinophils, neutrophils, platelets and dendritic cells also may have these and/or functionally related receptors.
Basophils can infiltrate sites of many immunologic or inflammatory processes, including IgE-associated late-phase reactions and sites of chronic allergic inflammation, often in association with eosinophils. Further, basophils can be involved in IgE independent mechanisms.
Basophils release several inflammatory mediators that have a role in the pathophysiology of allergic disease. The most commonly recognized inflammatory mediators are histamine and leukotriene C4 (LTC4), which cause smooth muscle contraction. It long has been thought, but not proved, that basophils release these substances during and/or after selectively infiltrating sites of allergic inflammation and thus contribute towards the symptoms of the “late phase response” (LPR). Basophils circulate in the blood under homeostatic conditions but will migrate into tissue during the LPR, which often follows acute allergic reactions. The exact mechanism of how they achieve this is not fully understood, in part due to the limited number of basophil studies that have resulted due to a lack of protocols to separate basophils from other effector cell populations.
Human basophils also release several other substances that are believed to possess inflammatory properties, although their exact role in allergic inflammation remains unclear. For example, basogranulin (which is defined by the monoclonal antibody BB1), a granule-specific highly basic protein secreted as a large complex (approximately 5×106 Da) by basophils, is secreted in vitro under the same conditions important for histamine release, including those occurring with both IgE-dependent and IgE-independent stimulation. Further, basophils also are believed to synthesize and secrete granzyme B (a serine protease).
In humans, basophils appear to be the prime early producers of the Th2-type cytokines IL-4 and IL-13, which perform several crucial functions in initiating and maintaining allergic responses. This putative immunomodulatory role of basophils is supported further by their ability to express CD40 ligand, which, together with IL-4 and IL-13, serve as inducers of B cell proliferation and class switching to IgE and IgG4. Moreover, human basophils are the main cellular source for rapid IL-4 generation, a mandatory requirement for the development of Th2 responses. Staining techniques have localized basophils in various tissues affected by allergic diseases. Some studies suggest that the interaction of basophils, T cells and B cells at these sites propagate pro-allergic immune responses. Additionally, basophil activation is not restricted to antigen-specific IgE crosslinking but can be caused in non-sensitized individuals by parasitic antigens, plant lectins and viral superantigens binding to non-specific IgEs. The presence of novel IgE-independent receptor targets that cause trafficking and Th2 cytokine release from basophils further underlines their potential role in innate as well as adaptive immunity.
Eosinophils
Eosinophils are primarily tissue-dwelling granulocytes that are recruited to sites of acute inflammation, and are seen most prominently in response to respiratory, gastrointestinal, and dermatologic allergens, as well as to generalized infection with helminthic parasites. Traditionally, functions of eosinophils focused singularly on their roles as end-stage “effector” cells, for example, in releasing their four granule cationic proteins and generating paracrine mediators of inflammation (such as ei-cosanoids). Studies have focused on eosinophils based in part on the recognition that eosinophils have distinct innate capacities to secrete differentially multiple preformed cytokines. Eosinophil-associated allergic inflammatory diseases notably occur in the airways and include nasal polyposis, allergic rhinoconjunctivitis and asthma. Eosinophils recruited into the mucosal airway tissues and secretions are positioned to encounter aeroallergens where it is thought they may assume a role as an antigen-presenting cell (APC). For example, in humans, blood eosinophils, which normally do not display MHC II proteins, can be induced to do so by stimulation with cytokines, including GM-CSF, IL-3, IL-4, IL-5 and interfereon-γ (IFN-γ). Moreover, human eosinophils recruited into the airways, as evidenced in the sputum of asthmatics and in lung lavages after allergen challenges, typically express MHC class II proteins. Unlike the gastroinstestinal tract where eosinophils normally are found and might be exposed to gut-derived antigens, eosinophils are not abundant in the normal lungs or airways. In contrast, recruitment of eosinophils into the upper and lower airways is a frequent concomitant of allergic inflammation. It is in this setting of allergic airways diseases that recruited eosinophils might function not simply as effectors of local inflammation, but also as “inflammatory” full-function antigen-presenting cells in processing and presenting airway antigens. In the context of allergic upper and lower airways diseases in which eosinophils are characteristically elicited, the capacity of eosinophils to serve as additional recruited “inflammatory” full-function APCs could be pertinent to antigen-elicited immune responses in the airways of those with often chronic, eosinophilic allergic diseases.
Neutrophils
Neutrophil leukocytes are crucial to both immunity and inflammation, and prolonged neutropenia (a decrease in the presence of neutrophils) leads to inevitable demise as a result of overwhelming infection. Neutrophils normally represent between 40% and 50% of the circulating leukocyte population, and they are easily recognized on a Wright's stained blood smear (a histologic stain that facilitates the differentiation of blood cell types) by their size, their characteristic multilobed nuclei, and the presence of fine stippling (representing granules throughout the cytoplasmic compartment). Primary and secondary granules contain distinct sets of their own proinflammatory mediators.
Neutrophils in the circulation are quiescent cells with only the potential to mediate a wide range of inflammatory activities. This potential is realized when neutrophils are activated. Neutrophils can be activated by a large number of specific agents, including, but not limited to, the following:
Activating AgentFunctionleukotriene B4 (LTB4)a chemoattractant that enhances adherence toendothelial cells and activates degranulationand NADPH oxidase activitycomplement fragmentchemoattractant that induces degranulation andC5aadherenceplatelet activatinginduces aggregation, adherence andfactor (PAF)degranulationhistamineinduces concentration-dependent changes inchemotaxis priming and degranulationinterferon-γ (IFN-γ)increases antibody-dependent cytotoxicity andpriminggranulocyte colony-increases antibody-dependent cytotoxicity andstimulating factorpriming, and enhances phagocytosis(G-CSF)granulocyte-macrophageinduces priming and stimulates maturationcolony-stimulating factorwithin the bone marrow(GM-CSF)IL-8chemoattractant that induces degranulation andNADPH oxidase activitytumor necrosis factor-αchemoattractant that induces priming, enhances(TFN-α)phagocytosis and antibody-dependentcytotoxicityfMet-Leu-Phechemoattractant that induces aggregation,degranulation and NADPH oxidase activity
As a group, these activating agents transmit signals to neutrophils via interaction with specific cell surface receptors, many of which interact with intracellular G proteins. G proteins catalyze the hydrolysis of guanosine triphosphate (GTP) to guanosine diphosphate (GDP) and inorganic phosphate, and initiate a series of events including activation of phospholipase C, initiation of calcium fluxes, and membrane depolarization. Once activated, neutrophils are able to adhere to endothelial cells, migrate through the endothelial barrier, and ingest and at least attempt to destroy pathogens, foreign bodies, and remnants of tissue damage. Neutrophils primed by brief exposure to activating agents (for example, but not limited to, endotoxin, IL-1, fMet-Leu-Phe, and GM-CSF) exhibit an enhanced response to subsequent stimuli. Both short-term (including changes in cell shape, oxidative and phagocytic capacity) and long-term (prolonged cell viability) responses to priming agents have been observed.
Neutrophils contain both primary and secondary granules each with distinct effector proteins. Major components of primary granules (azurophil) include myeloperoxidase (which converts hydrogen peroxide generated by NADPH oxidase and hydrochloric acid to hypochlorous acid), defensins (having antibacterial activity), bacterial permeability-increasing protein (BPI; having antibacterial activity), cathepsin G (antibacterial activity), lysozyme (which digests the peptidoglycan component of most bacterial cell walls), elastase, alkaline phosphatase, proteinase 3, β-glucuronidase, phospholipases A2, C and D, and α-mamosidase. The major secondary components include lactoferrin (an iron-binding protein with some antibacterial activity), gelatinase, collagenase, vitamin B12-binding protein, lysozyme, cytochrome b558, fMLP receptor, integrins (CD11b/CD18, CD11c/CD18), complement receptor 3 (CR3), histaminase, and plasminogen activator.
2. Agranulocytes
Agranulocytes (mononuclear leukocytes) are characterized by the apparent absence of granules in their cytoplasm. These cells contain azurophilic granules, which are lysosomes. Agranulocytes include lymphocytes, monocytes and macrophages.
Isolating Blood Granulocyte Responses
Subsets of granulocytes can be isolated from blood by a variety of physical techniques, such as, for example, density gradient centrifugation. Magnetic particles associated with monoclonal antibodies can be specifically bound to subsets of granulocytes and “stuck” temporarily to magnetic material to “positively isolate” the subsets. Alternatively, contaminating subsets can be removed by binding magnetic beads to them. However, in all cases, the manipulations and time constraints inherent in physical separation methods may initiate non-specific activation of the granulocytes. This has been demonstrated to occur even with very short and relatively gentle manipulations.
The activation of the granulocytes during these isolation procedures, which also can include flow cytometry cell sorting methods, is demonstrable by the appearance of CD11b, CD63, CD203, CD66b and other activation markers on the various granulocyte subpopulations. The presence of these markers is detectable by flow cytometry analysis of the isolated subpopulations.
Flow Cytometry
Flow cytometry, a technique that may be used for counting and examining cells, allows simultaneous multiparametric analysis of the physical and/or chemical characteristics of each individual cell. Briefly, a beam of light (usually laser light) of a single wavelength is directed onto a hydrodynamically-focused stream of fluid. A number of detectors are aimed at the point where the stream passes through the light beam: one in line with the light beam (Forward Scatter (FSC)), several perpendicular to it (Side Scatter (SSC)), and one or more fluorescence detectors. Each suspended cell (from 0.2 μm to 150 μm) passing through the light beam scatters the light in some way, and fluorescent molecules (naturally occurring or as part of an attached label or dye) may be excited into emitting light at a longer wavelength than the light source. This combination of scattered and fluorescent light is recorded by the detectors. The FSC correlates with the cell volume; SSC depends upon the inner complexity of the cell (i.e., shape of the nucleus, type of cytoplasmic granules, etc.). The data generated by flow cytometers may be plotted as a histogram. The regions on these plots can be separated sequentially based on fluorescence intensity by creating a series of subset extractions (“gates”). Specific gating protocols have been developed for diagnostic and clinical purposes.
Fluorescence activated cell sorting (FACS) provides a method of sorting a heterogeneous mixture of cells into two or more containers, a single cell at a time, based upon the specific light scattering and fluorescent characteristics of each cell. Briefly, the cell suspension is entrained in the center of a narrow, rapidly flowing stream of liquid and the flow is arranged such that there is a large separation between cells relative to their diameter. The stream of individual cells passes through a fluorescence detector, and an electrical charge is assigned to each cell (based on the cell's fluorescence) just as the stream is broken into individual drops (usually via vibration) such that there is a low probability of more than one cell per droplet. Each charged droplet (containing an individual cell) may be sorted, via electrostatic deflection, into separate containers.
The surfaces of all cells in the body are coated with specialized protein receptors that selectively can bind or adhere to other signaling molecules. These receptors and the molecules that bind to them are used for communicating with other cells and for carrying out proper cell functions in the body. Each cell type has a certain combination of receptors (or surface markers) on its surface that makes it distinguishable from other kinds of cells. Cells may be fluorescently labeled, i.e., a reactive derivative of a fluorophore may be covalently attached to a cell. The most commonly used labeled molecules are antibodies; their specificity towards certain surface markers on a cell surface allows for more precise detection and monitoring of particular cells. The fluorescence labels that can be used will depend upon the lamp or laser used to excite the fluorochromes and on the detectors available. For example, when a blue argon laser (448 nm) is used, fluorescent labels used may include, but are not limited to, fluorescein isothiocyanate (FITC), Alexa Fluor® 488, green fluorescent protein (GFP), carboxyfluorescein (CFSE), carboxyfluorescein diacetate succinimidyl ester (CFDA-SE), DyLight® 488 (Dyomics), phycoerythrin (PE), propidium iodide (PI), peridinin chlorophyll protein (PerCP), PerCP-Cy™ 5.5, PE-AlexaFluor 700, PE-Cy™ 5; PE-Cy™ 5.5, PE-AlexaFluor® 750 and PE-Cy™ 7; when a red diode laser (635 nm) is used, fluorescent labels used may include, but are not limited to, allophycocyanin (APC), APC-Cy™ 7, APC-eFluor® 780, AlexFluor® 700, Cy™ 5, and Draq-5; when a violet laser is used (405 nm), fluorescent labels may include, but are not limited to, Pacific Orange™, amine aqua, Pacific Blue™, 4′-6-diamidino-2-phenylindole (DAPI), AlexFluor® 405, and eFluor® 450.
The development of flow-cytometry based approaches to the identification of activation markers and intracellular markers, via measurement of enzymatic and surface marker profiles, has allowed for accelerated association of surface topologies with disease states. Studies that involve the triggering of cells to respond to environmental stimuli, such as an allergen or drug action, and the activation phenotypes associated with such agitation, allow for clearer resolution of the underlying activation states and provide for more distinct classification of allergic disease outcomes. Allergy is a dynamic event, and as such, static views of basal states would be considered insufficient for determination of an activated state, therefore rendering correlations to clinical outcomes less meaningful. Fractionation of cell populations with flow cytometry is well suited to address activation markers and intracellular markers in the context of allergic diseases, because it can simultaneously discern multiple surface markers within complex cellular populations.
Cluster of Differentiation
The cluster of differentiation (CD) system is a protocol used for the identification of cell surface molecules present on white blood cells. CD molecules can act in numerous ways, often acting as receptors or ligands; by which a signal cascade is initiated, altering the behavior of the cell. Some CD proteins do not play a role in cell signaling, but have other functions, such as cell adhesion. Generally, a proposed surface molecule is assigned a CD number once two specific monoclonal antibodies (mAb) are shown to bind to the molecule. If the molecule has not been well-characterized, or has only one mAb, the molecule usually is given the provisional indicator “w.”
The CD system nomenclature commonly used to identify cell markers thus allows cells to be defined based on what molecules are present on their surface. These markers often are used to associate cells with certain immune functions. While using one CD molecule to define populations is uncommon, combining markers has allowed for cell types with very specific definitions within the immune system. There are more than 350 CD molecules identified for humans.
CD molecules are utilized in cell sorting using various methods, including flow cytometry. Cell populations usually are defined using a “+” or a “−” symbol to indicate whether a certain cell fraction expresses or lacks a CD molecule. For example, a “CD34+, CD31−” cell is one that expresses CD34, but not CD31. Table 1 shows commonly used markers employed by skilled artisans to identify and characterize differentiated white blood cell types:
Type of CellCD MarkersStem cellsCD34+, CD31−All leukocyte groupsCD45+GranulocyteCD45+, CD15+MonocyteCD45+, CD14+T lymphocyteCD45+, CD3+T helper cellCD45+, CD3+, CD4+Cytotoxic T cellCD45+, CD3+, CD8+B lymphocyteCD45+, CD19+ or CD45+, CD20+ThrombocyteCD45+, CD61+Natural killer cellCD16+, CD56+, CD3−
CD molecules used in defining leukocytes are not exclusively markers on the cell surface. Most CD molecules have an important function, although only a small portion of known CD molecules have been characterized. For example, there are over 350 CD for humans identified thus far.
CD3 (TCR complex) is a protein complex composed of four distinct chains. In mammals, the complex contains a CD3γ chain, a CD3δ chain, and two CD3ε chains, which associate with the T cell receptor (TCR) and the ζ-chain to generate an activation signal in T lymphocytes. Together, the TCR, the ζ-chain and CD3 molecules comprise the TCR complex. The intracellular tails of CD3 molecules contain a conserved motiff known as the immunoreceptor tyrosine-based activation motif (ITAM), which is essential for the signaling capacity of the TCR. Upon phosphorylation of the ITAM, the CD3 chain can bind ZAP70 (zeta associated protein), a kinase involved in the signaling cascade of the T cell.
CD14 is a cell surface protein expressed mainly by macrophages and, to a lesser extent, neutrophil granulocytes. CD14+ cells are monocytes that can differentiate into a host of different cells; for example, differentiation to dendritic cells is promoted by cytokines such as GM-CSF and IL-4. CD14 acts as a co-receptor (along with toll-like receptor (TLR) 4 and lymphocyte antigen 96 (MD-2)) for the detection of bacterial lipopolysaccharide (LPS). CD14 only can bind LPS in the presence of lipopolysaccharide binding protein (LBP).
CD15 (3-fucosyl-N-acetyl-lactosamine; stage specific embryonic antigen 1 (S SEA-1)) is a carbohydrate adhesion molecule that can be expressed on glycoproteins, glycolipids and proteoglycans. CD15 commonly is found on neutrophils and mediates phagocytosis and chemotaxis.
CD16 is an Fc receptor (FcγRIIIa and FcγRIIIb) found on the surface of natural killer cells, neutrophil polymorphonuclear leukocytes, monocytes and macrophages. Fc receptors bind to the Fc portion of IgG antibodies.
CD19 is a human protein expressed on follicular dendritic cells and B cells. This cell surface molecule assembles with the antigen receptor of B lymphocytes in order to decrease the threshold for antigen receptor-dependent stimulation. It generally is believed that, upon activation, the cytoplasmic tail of CD19 becomes phosphorylated, which allows binding by Src-family kinases and recruitment of phosphoinositide 3 (PI-3) kinases.
CD20 is a non-glycosylated phosphoprotein expressed on the surface of all mature B-cells. Studies suggest that CD20 plays a role in the development and differentiation of B-cells into plasma cells. CD20 is encoded by a member of the membrane-spanning 4A gene family (MS4A). Members of this protein family are characterized by common structural features and display unique expression patterns among hematopoietic cells and nonlymphoid tissues.
CD31 (platelet/endothelial cell adhesion molecule; PECAM1) normally is found on endothelial cells, platelets, macrophages and Kupffer cells, granulocytes, T cells, natural killer cells, lymphocytes, megakaryocytes, osteoclasts and neutrophils CD31 has a key role in tissue regeneration and in safely removing neutrophils from the body. Upon contact, the CD31 molecules of macrophages and neutrophils are used to communicate the health status of the neutrophil to the macrophage.
CD34 is a monomeric cell surface glycoprotein normally found on hematopoietic cells, endothelial progenitor cells, endothelial cells of blood vessels, and mast cells. The CD34 protein is a member of a family of single-pass transmembrane sialomucin proteins and functions as a cell-cell adhesion factor. Studies suggest that CD34 also may mediate the attachment of stem cells to bone marrow extracellular matrix or directly to stromal cells.
CD45 (protein tyrosine phosphatase, receptor type, C; PTPRC) cell surface molecule is expressed specifically in hematopoietic cells. CD45 is a protein tyrosine phosphatase (PTP) with an extracellular domain, a single transmembrane segment, and two tandem intracytoplasmic catalytic domains, and thus belongs to receptor type PTP. Studies suggest it is an essential regulator of T-cell and B-cell antigen receptor signaling that functions by direct interaction with components of the antigen receptor complexes, or by activating various Src family kinases required for antigent receptor signaling. CD45 also suppresses JAK kinases, and thus functions as a regulator of cytokine receptor signaling. The CD45 family consists of multiple members that are all products of a single complex gene. Various known isoforms of CD45 include: CD45RA, CD45RB, CD45RC, CD45RAB, CD45RAC, CD45RBC, CD45RO, and CD45R (ABC). Different isoforms may be found on different cells. For example, CD45RA is found on naïve T cells and CD45RO is found on memory T cells.
CD56 (neural cell adhesion molecule, NCAM) is a homophilic binding glycoprotein expressed on the surface of neurons, glia, skeletal muscle and natural killer cells. It generally is believed that NCAM has a role in cell-cell adhesion, neurite outgrowth, and synaptic plasticity. There are three known main isoforms of NCAM, each varying only in their cytoplasmic domains: NCAM-120 kDA (glycosylphopharidylinositol (GPI) anchored); NCAM-140 kDa (short cytoplasmic domain); and NCAM (long cytoplasmic domain). The different domains of NCAM have different roles, with the Ig domains being involved in homophilic binding to NCAM, and the fibronection type III (FNIII) domains being involved in signaling leading to neurite outgrowth.
CD66b ((CGM1); CD67, CGM6, NCA-95) is a glycosylphosphatidylinositol (GPI)-linked protein that is a member of the immunoglobulin superfamily and carcinoembryonic antigen (CEA)-like subfamily. CD66b, expressed on granulocytes, generally is believed to be involved in regulating adhesion and activation of human eosinophils.
Human leukocyte antigen (HLA)-DR is a major histocompatibility complex (MHC) class II cell surface receptor. HLA-DR commonly is found on antigen-presenting cells such as macrophages, B-cells, and dendritic cells. This cell surface molecule is a αβ heterodimer with each subunit containing 2 extracellular domains: a membrane spanning domain and a cytoplasmic tail. Both the α a and β chains are anchored in the membrane. The complex of HLA-DR and its ligand (a peptide of at least 9 amino acids) constitutes a ligand for the TCR.
Integrins are receptors that mediate attachment between a cell and the tissues surrounding it and are involved in cell-cell and cell-matrix interactions. In mammals, 18α and 8β subunits have been characterized. Both α and β subunits contain two separate tails, both of which penetrate the plasma membrane and possess small cytoplasmic domains.
Integrin αM (ITGAM; CD11b; macrophage-1 antigen (Mac-1); complement receptor 3 (CR3)) is a protein subunit of the heterodimeric integrin αMβ2 molecule. The second chain of αMβ2 is the common integrin β2 subunit (CD18). αMβ2 is expressed on the surface of many leukocytes including monocytes, granulocytes, macrophages and natural killer cells. It generally is believed that αMβ2 mediates inflammation by regulating leukocyte adhesion and migration. Further, αMβ2 is thought to have a role in phagocytosis, cell-mediated cytotoxicity, chemotaxis and cellular activation, as well as being involved in the complement system due to its capacity to bind inactivated complement component 3b (iC3b). The ITGAM subunit of integrin αMβ2 is involved directly in causing the adhesion and spreading of cells, but cannot mediate cellular migration without the presence of the β2 (CD18) subunit.
CD61 (integrin β3; platelet glycoprotein IIIa; ITGB3) is a cell surface protein composed of an α-chain and a β-chain. A given chain may combine with multiple partners resulting in different integrins. CD61 is found along with the α IIb chain in platelets and is known to participate in cell adhesion and cell-surface mediated signaling.
CD63 (LAMP-3; ME491; MLA1; OMA81H) is a cell surface glycoprotein of the transmembrane 4 superfamily (tetraspanin family). Many of these cell surface receptors have four hydrophobic domains and mediate signal transduction events that play a role in the regulation of cell development, activation, growth and motility. CD63 forms complexes with integrins and may function as a blood platelet activation marker. It generally is believed that the sensitivity and specificity of measuring the upregulation of CD63 alone, or as part of a combination, is not specific enough to serve as a diagnostic marker for the diagnosis of IgE mediated allergy.
CD123 is the 70 kD transmembrane a chain of the cytokine interleukin-3 (IL-3) receptor. Alone, CD123 binds IL-3 with low affinity; when CD123 associates with CDw131 (common β chain), it binds IL-3 with high affinity. CD123 does not transduce intracellular signals upon binding IL-3 and requires the β chain for this function. CD123 is expressed by myeloid precursors, macrophages, dendritic cells, mast cells, basophils, megakaryocytes, and some B cells CD123 induces tyrosine phosphorylation within the cell and promotes proliferation and differentiation within the hematopoietic cell lines.
CD203c (ectonucleotide pyrophosphatase/phosphodiesterase 3; ENPP3) is an ectoenzyme constitutively and specifically expressed on the cell surface and within intracellular compartments of basophils, mast cells, and precursors of these cells. CD203c detection by flow cytometry has been used to specifically identify basophils within a mixed leukocyte suspension, since its expression is unique to basophils among the cells circulating in blood. The expression of CD203c is both rapidly and markedly upregulated following IgE-dependent activation. However, as with CD63, it is generally believed that the sensitivity and specificity of measuring the upregulation of CD203c alone, or as part of a combination, is not specific enough to serve as a diagnostic marker for the diagnosis of IgE mediated allergy. Further, the exact role of CD203c in basophil biology is unknown.
CD294 (G protein-coupled receptor 44; GPR44; CRTh2; DP2) is an integral membrane protein. This chemoattractant receptor homologous molecule is expressed on T helper type-2 cells. The transmembrane domains of these proteins mediate signals to the interior of the cell by activation of heterotrimeric G proteins that in turn activate various effector proteins that ultimately result a physiologic response.
Need for Methodologies and Assays
The analysis of subpopulations of white blood cells remains of particular interest for the evaluation of immune system disorders, especially allergic diseases. Basophils and eosinophils, both important components of the allergic response, and the activation phenotypes they display during a response to an environmental stimuli, provide an important focus of study of allergy treatment, detection and classification.
Many conventional approaches of monitoring cell activation make use of potentially biased extended in vitro-cultured cells. Further, many conventional methods of counting white blood cells, such as basophils and eosinophils, are based on granule-staining with subsequent manual or automated counting. These methods are time consuming and lack specificity towards cell activation and distinguishing between live cells and dead cells. Conventional purification kits remain based on density gradient separation followed by cell sorting; these methods typically fail to provide pure cell samples and eliminate contamination.
Further, many assays for allergy to foods have disadvantages. Blood tests for specific immunoglobulin levels lack high specificity while the “gold standard” for identifying a food allergen to which a patient is allergic is a costly, double-blind, placebo-controlled in vivo food challenge (DBPCFC). The DBPFC requires an often lengthy hospitalization and places the patient at risk for induction of anaphylaxis, as these skin tests may be associated with anaphylaxis. These safety concerns impair the ability to identify offending allergens. Furthermore, clinical studies on food allergy are difficult to conduct given the need for skin tests and DBPCFC. Many assays designed to aid in the diagnosis of food allergy are technically complex, lack adequate sensitivity, are non-specific and require prolonged time periods.
The described invention addresses the inadequacies of these methods. It provides the ability to monitor cell activation in patient samples such as whole blood, more specifically white blood cells such as basophils and eosinophils, therein, and allows for measuring activation ex vivo. Further, it allows for physiologic interpretations in situations, for instance, where immune action depends on natural context, for close monitoring of activation states within patients who have an allergic disease, and for correlation of disease state and treatment. Additionally, the described invention provides a method of isolating basophils from stimulated or unstimulated blood based that is independent of the IgE/High affinity IgE receptor on the basophil cell surface.